Note pubs.acs.org/jnp
Asteltoxins from the Entomopathogenic Fungus Pochonia bulbillosa 8‑H-28 Hayamitsu Adachi,*,† Hiroyasu Doi,† Yuichi Kasahara,† Ryuichi Sawa,† Kaori Nakajima,† Yumiko Kubota,† Nobuo Hosokawa,† Ken Tateishi,‡ and Akio Nomoto† †
Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan
‡
S Supporting Information *
ABSTRACT: New asteltoxins C (3) and D (4) were found in the extract of the entomopathogenic fungus Pochonia bulbillosa 8-H28. Compound 2, which was spectroscopically identical with the known asteltoxin B, was isolated, and structural analysis led to a revision of the structure of asteltoxin B. Compounds 2 and 4 have a novel tricyclic ring system connected to a dienyl α-pyrone structure. Compound 3 has a 2,8-dioxabicyclo[3.3.0]octane ring similar to that of asteltoxin (1). Compound 3 showed potent antiproliferative activity against NIAS-SL64 cells derived from the fat body of Spodoptera litura larvae, while 2 and 4 were inactive.
A
structure of asteltoxin has been a target for organic synthesis.3,13−15 Asteltoxin B16 was isolated from the fungus Aspergillus sp. SCSGFA 0076. The ethyl group at C-3 and the hydroxy group at C-4 are in the β-configuration, thus differing from the stereochemistry in asteltoxin. The double bond between C-9 and C-10 is oxygenated to form an epoxide. In the course of screening for cytotoxic substances against NIAS-SL64 cells17 derived from the fat body of Spodoptera litura larvae, asteltoxin1 and a novel compound (3) were isolated from the culture broth of the fungus Pochonia bulbillosa 8-H-28. The P. bulbillosa strain 8-H-28 was isolated from the fruiting body of Elaphocordyceps capitata collected in Hanno, Saitama Prefecture, Japan. Similar strains of Pochonia genus are known to produce other substances; for example, P. suchlasporia var. suchlasporia TAMA 87 produces pochonicine, a polyhydroxylated pyrrolidine alkaloid that functions as a potent β-N-acetylglucosaminidase inhibitor,18 and P. chlamydosporia produces aurovertin-type nematode toxins.19 An ethyl acetate extract of the culture broth of P. bulbillosa 8H-28 inhibited the growth of NIAS-SL64 cells. Analysis of the active fractions led to the identification of asteltoxin and 3. The main asteltoxin produced by P. bulbillosa 8-H-28 strain was asteltoxin. Compound 3 was obtained as a yellow solid. The molecular formula was determined to be C22H28O7 based on HRESIMS and NMR data. The optical rotation of 3 showed a positive
steltoxin (1, Figure 1), a metabolite of the fungus Aspergillus stellatus Cruiz, is a mycotoxin1 that shows
Figure 1. Structures of asteltoxin (1), asteltoxins B (revised structure) (2), C (3), and D (4), and literature asteltoxin B.16
inhibitory activity against Escherichia coli BF1-ATPase.2 The structure of 1 was determined by spectroscopic methods and single-crystal X-ray analysis, and its absolute configuration was subsequently established by partial synthesis starting with (R)isopropylidene glyceraldehyde.3 Asteltoxin is related to citreoviridin4−6 and aurovertin,7−12 all having a trienic αpyrone structure. However, unlike citreoviridin and aurovertin, asteltoxin has a unique, highly functionalized 2,8dioxabicyclo[3.3.0]octane ring containing a quaternary carbon embedded in an array of six stereogenic centers. This unique © XXXX American Chemical Society and American Society of Pharmacognosy
Received: August 30, 2014
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DOI: 10.1021/np500676j J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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Figure 2. (a) HMBC and COSY correlations of 2 (revised structure of asteltoxin B), (b) ROE correlations of 2 (revised structure of asteltoxin B), (c) HMBC and COSY correlations of 3, (d) ROE correlations of 3, (e) HMBC and COSY correlations of 4, (f) ROE correlations of 4.
value of [α]27D +11.3 (c 0.1, MeOH), similar to that of asteltoxin ([α]20D +20, c 1.15, MeOH).1 Compound 3 had a UV spectrum similar to that of asteltoxin (2: UV (MeOH) λmax (log ε) 364 (4.35), 274 (4.33), 269 (4.32); 1: 367 (4.51), 272.5 (4.46), 267 (4.46)).1 The IR spectrum of 3 showed two characteristic absorptions of CO stretching at 1693 and 1621 cm−1 consistent with the presence of an α-pyrone unit. The planar structure of 3 was established from analysis of 1H, 13 C, HMQC, HMBC, and COSY data (Figures 2c and S9−S14, Table 1). The 1H and 13C NMR spectra of 3 were similar to those of asteltoxin1 except for the methyl group at C-2 of 3. The presence of a 2,8-dioxabicyclo[3.3.0]octane ring structure was supported by the observed HMBC correlations of H-1/C3, H-5/C-2, C-4, C-7, C-20, H-20/C-3, C-4, C-5, C-6, and H19/C-2, C-3, C-4. The relative configuration of 3 was determined by analyzing the 1D ROE spectra as shown in Figures 2d and S15−S16. Key ROE correlations were observed between H-2/H-19 and H-19/HO-6 and H-8, illustrating the preferable conformation where H-2, H-19, and HO-6 are oriented on the same face of the 2,8-dioxabicyclo[3.3.0]octane ring. The correlations of H-5/HO-3, H-7 and H-20 and H-20/ HO-3, H-6, and H-7 indicated that H-1, HO-3, H-20, H-5, H-6, and H-7 are on the opposite face of the ring. The relative configuration of 3 would be identical with that of asteltoxin, and the absolute configuration of 3 is presumed to be analogous to that of asteltoxin based on the similarity of
optical rotations of asteltoxin and 3. The 2,8dioxabicyclo[3.3.0]octane ring structure connected by triene with α-pyrone was established by 2D NMR and by comparison with the published data for asteltoxin. Asteltoxin and 3 slowly decomposed to a complex mixture in solution during purification steps through unclear mechanisms. Compound 3 was named asteltoxin C. Based on the similarity of their NMR spectra to those of asteltoxin and 3, compounds 2 and 4 were also identified, respectively, although compounds 2 and 4 did not exhibit antiproliferative activity against NIAS-SL64 cells. 1H and 13C NMR data of 2 (Table 1) were the same as those of asteltoxin B.16 The IR peaks of 2 at 3435, 2935, 1697, 1631, 1544, 1456, 1405, 1252, 1092, and 1063 were similar to those of asteltoxin B.16 However, the optical rotation of 2 differed slightly from that of asteltoxin B: [α]25D +93.1 (c 0.1, MeOH); asteltoxin B: [α]20D +76 (c 0.1, MeOH). Also, the UV spectrum of 2 showed λmax (log ε) at 343 (4.30) and 248 (4.45), different from those in the UV spectrum of asteltoxin B (λmax (log ε) 330 (4.04) and 288 (3.98)).16 Even though compound 2 differed from asteltoxin B in terms of its optical rotation and UV spectrum, the consistency of NMR spectra, especially the 13C NMR spectrum, is highly reliable. Therefore, the structure of 2 would be identical to that of asteltoxin B. The planar structure of 2 was established from 1H, 13C, HMQC, HMBC, and COSY spectra (Figures 2a and S1−S6, B
DOI: 10.1021/np500676j J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 1H and 13C NMR Data for Compounds 2 (Revised Structure of Asteltoxin B), the Literature Asteltoxin B, 3, and 4a asteltoxin B lit. data16
asteltoxin B revised (2) position
δC
1 2
11.3 22.3
3
90.6
4-OH 4 5 6 7
δH (mult J in Hz) 1.06 1.52 1.57 4.09
(t 7.5) (m) (m) (dd 3.7, 9.2)
1.38 (s) 80.5 62.5 113.8 87.3
8 9-OH 9
86.2
10
5.47 (s) 4.27 (d 4.1)
position
δC
1 2
11.5 22.4
1.06 (t 7.43) 1.58 (m)
3
90.8
4-OH 4 5 6 7
80.6 62.7 113.9 87.4
asteltoxin C (3)
δH (mult J in Hz)
position
δC
1
13.7
1.20 (d 6.4)
4.10 (dd 3.7, 9.05)
2
83.6
4.58 (q 6.3)
2
84.4
4.36 (t 6.4)
81.0
3 3-OH 4 5 6
80.5
5.47 (s) 4.27 (d 4.05)
3 3-OH 4 5 6 6-OH 7 8
82.8 128.8
1.50 (s) 61.9 111.8 78.5
4.81 (t 4.4)
77.2
3.91 (brs)
83.3
10
83.4
4.38 (t 7.0)
9
134.1
11
136.1
5.98 (dd 6.6, 15.2)
11
136.2
10
12
131.0
6.44 (dd 11.2, 15.2)
12
131.2
13
134.6
7.15 (dd 11.1, 15.0)
13
134.8
14 15 16 17 18 19 20 21 22 23
120.3 154.0 108.4 170.5 89.1 163.5 19.0 14.9 8.9 56.2
6.39 (d 15.2)
14 15 16 17 18 19 20 21 22 23
120.5 154.1 108.6 170.7 89.3 163.8 19.1 15.1 9.1 56.4
5.98 (dd 6.5, 15.2) 6.47 (dd 11.1, 15.2) 7.17 (dd 11.1, 15.1) 6.39 (d 15.2)
(s) (s) (s) (s)
δH (mult J in Hz)
1.2 (d 6.3)
86.3
1.39 1.11 1.96 3.83
δC
12.8
8 9-OH 9
5.51 (s)
position
1
4.80 (t 4.4) 2.73 (d 11.2) 3.91 (ddd 4.7, 7.7, 12.2) 4.37 (t 7.2)
77.0
δH (mult J in Hz)
asteltoxin D (4)
5.51 (s) 1.39 1.11 1.96 3.83
(s) (s) (s) (s)
5.29 3.74 1.69 4.75 5.84
(s) (dd 3.0, 4.2) (d 4.3) (m) (dd 4.8, 15.3)
1.38 (s) 62.3 113.9 87.3
5.47 (s) 4.28 (d 4.0)
7 8 8-OH
86.2 77.2
4.79 (t 4.3) 3.91 (m) 2.67 (d 10.7)
9
83.2
136.1
6.67 (ddd 1.6, 11.0, 15.4) 6.51 (dd 11.0, 14.9)
10
136.0
11
132.9
6.41 (dd 11.1, 15.1)
11
131.0
12
135.3
7.19 (dd 11.1, 15.1)
12
134.6
13 14 15 16 17 18 19 20 21 22
120.3 154.1 108.4 170.5 89.0 163.6 17.4 16.3 8.9 56.2
6.39 (d 15.1)
13 14 15 16 17 18 19 20 21 22
120.3 154.0 108.4 170.5 89.1 163.5 18.4 15.2 8.9 56.2
4.38 (dd 6.2, 6.9) 5.97 (dd 6.5, 15.4) 6.45 (dd 11.1, 15.2) 7.14 (dd 11.2, 15.1) 6.39 (d 15.1)
5.51 (s) 1.36 1.21 1.98 3.83
(s) (s) (s) (s)
5.51 (s) 1.36 1.13 1.96 3.83
(s) (s) (s) (s)
a 1 H (600 MHz) and 13C (150 MHz) NMR spectra for 2−4 were measured in CDCl3. Chemical shifts are expressed in ppm with TMS as the internal standard.
NMR spectrum of asteltoxin B (see Supporting Information in ref 16), although this 1H signal was not assigned for structure determination. This 1H signal is very close to the 1H signal (2.73 ppm) of the hydroxy group at C-9 of 2. Thus, these NMR data clarified that the literature asteltoxin B does not have an epoxide at C-9 and C-10. Thus, the structure of asteltoxin B should be revised as possessing the tricyclic ring system as shown in compound 2. The relative configuration of the tricyclic ring system of 2 was elucidated by ROE correlations (Figures 2b and S7 and S8). A characteristic ROE correlation of 2 was observed between H-3 and H-10, which verified the stereochemistry at a sequence of carbon atoms from C-3 to C-10, illustrating the cage-like structure of the tricyclic ring system. Comparing the stereochemistry of the 2,8-dioxabicyclo[3.3.0]octane ring unit between 2 and the literature asteltoxin B, stereochemistries at C-3 and C-4 are opposite. Although the ROE data of the literature asteltoxin B show the correlation between H-3 and H10, this correlation would not support the stereochemistry at C3 and C-4 as drawn in the paper16 due to steric difficulties. If this correlation is valid, the stereochemistry at C-3 and C-4 would be opposite. Taken together, the structure of asteltoxin B was revised as that of compound 2. The stereochemistry of 2 at
Table 1). The tricyclic ring system was elucidated from the key HMBC correlation between H-7/C-7 and C-10/H-10, as shown in Figure 2a, indicating that C-7 is connected to C-10 via an ether oxygen. In other words, the proposed structure has the tetrahydrofuran ring fused at C-7 and C-8, in which the hydroxy group and ether oxygen are substituted at C-9 and C10, respectively. In addition, the 1H signal (2.73 ppm) of the hydroxy group at C-9 was observed and spin-coupled with H-9, indicating that the C-9 of 2 would be substituted with a hydroxy group, not the epoxy ether oxygen as in the literature asteltoxin B. The structural unit at C-7−C-10 of 2 was different from that of the literature asteltoxin B, having the epoxide at C9 and C-10. Although the structure of the literature asteltoxin B possesses a bicyclic ring system connected to an epoxide at C-9 and C-10, there are inconsistencies between the reported NMR data of asteltoxin B and its structure. The 13C chemical shift values, 77.2 and 83.4 ppm at C-9 and C-10 (Table 1: the literature asteltoxin B), respectively, would be too large for an epoxide carbon (ref 20: typically 40−65 ppm for a substituted epoxide carbon), suggesting typical oxygen substitutions at C-9 and C-10 without the strain of an epoxide. A hydroxy group or ether oxygen would be a possible substituent at C-9 and C-10. In addition, the 1H signal (∼2.6 ppm) was observed in the 1H C
DOI: 10.1021/np500676j J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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were seeded in 96-well microplates at a density of 5 × 103 cells in 100 μL of MGM-450 medium per well in the presence of various concentrations of asteltoxins or beauvericin. After a 24 h incubation, MTS was added to each well, and the plate was further incubated for 4 h. The absorbance at 490 nm was measured using an ARVO-SX 1420 multilabel counter (PerkinElmer, Waltham, MA, USA). Beauvericin was used as positive control compound, and DMSO utilized to dissolve asteltoxins and beauvericin was used as negative control. Fermentation. A slant culture of P. bulbillosa 8-H-28 was used for inoculation of the fungus into 500 mL Erlenmeyer flasks. Each flask contained 100 mL of medium [3% glucose, 0.5% Bacto Peptone (Becton, Dickinson and Company), 0.3% yeast extract (Becton, Dickinson and Company), 0.03% KH2PO4, 0.03% K2HPO4, and 0.03% MgSO4·7H2O in deionized water, with pH adjusted to 6.0 before sterilization]. After inoculation of P. bulbillosa 8-H-28 into the medium, the flasks (420 in total) were shaken on a rotary shaker at 180 rpm for 14 days at 25 °C. Acetone (100 mL) was added to the medium, and the mixture was shaken on a rotary shaker at 180 rpm overnight at 10 °C. Isolation of Asteltoxins. The combined mixture was filtered, and the acetone was removed by evaporation to give an aqueous solution. The aqueous solution was extracted with hexane and then with ethyl acetate. The ethyl acetate layer was dried over MgSO4 and filtered. The filtrate was evaporated, leaving a brown solid (13.3 g). The solid was subjected to repeated rounds of silica gel column chromatography (Cica silica gel 60N, Kanto Chemical Co. Inc., Tokyo, Japan) and eluted with a mixture of CHCl3 and MeOH (20:1). The active fractions were combined, and the solvent was evaporated to give another solid. The solid was subjected to reversed-phase HPLC on a Shiseido Capcell Pak C18 MGII column (20 mm × 250 mm, 5 μm) eluted with 32−35% acetonitrile in water to give partially purified substances. Also fractions including substances that showed similar NMR spectra to those of active fractions were purified. These substances were further subjected to reversed-phase HPLC on a Shiseido Capcell Pak C18 SG120 column (30 mm × 250 mm, 5 μm) eluted with 22−23% acetonitrile in water, yielding compounds 1 (190 mg), 2 (180 mg), 3 (10 mg), and 4 (15 mg). Asteltoxin B (revised structure) (2): yellow powder; [α]25D +93.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 343 (4.30), 248 (4.45); IR (KBr) νmax 3435, 2935, 1697, 1631, 1544, 1456, 1405, 1252, 1092, 1063 ; 1H and 13C NMR (Table 1); HRESIMS m/z 435.2007 [M + H]+ (calcd for C23H31O8, 435.2013). Asteltoxin C (3): yellow powder; [α]27D +11.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 364 (4.35), 274 (4.33), 269 (4.32); IR (KBr) νmax 3430, 2930, 1693, 1621, 1539, 1455, 1405, 1251, 1003; 1H and 13 C NMR (Table 1); HRESIMS m/z 405.1908 [M + H]+ (calcd for C22H29O7, 405.1908). Asteltoxin D (4): yellow powder; [α]27D +127.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 343 (4.48), 248 (4.66); IR (KBr) νmax 3425, 2935, 1695, 1630, 1543, 1456, 1406, 1253, 994; 1H and 13C NMR (Table 1); HRESIMS m/z 421.1859 [M + H]+ (calcd for C22H29O8, 421.1857).
C-3 and C-4 was opposite to that of the literature asteltoxin B, and 2 possesses a tricyclic ring system with a hydroxy group at C-9 as shown in Figure 1. Compound 4 was also obtained as a yellow solid. Spectroscopic data (1H, 13C, HMQC, HMBC, COSY, and ROE) are consistent with the structure in which the ethyl group at C-3 of 2 is replaced with a methyl group (Figure 2e and f, S17−S24, Table 1). Compound 4 was named asteltoxin D. The structural difference between compounds 1/2 and 3/4 is the replacement of the ethyl group with a methyl group. As it was demonstrated that the terminal methyl group at C-1 of 1 comes from methionine in biosynthesis by the enhancement of the 13C NMR signal at C-1 from a feeding experiment of (2S)[methyl-13C]methionine,21 it seems reasonable biosynthetically to have 2 and 3 as non-methylated versions of 1 and 4. Antiproliferative activities of new asteltoxins against NIASSL64 cells were evaluated and compared with the cytotoxicity of beauvericin against NIAS-SL64 cells. Beauvericin, a depsipeptide insecticidal antibiotic, is known to exhibit moderate cytotoxic activities against MCF-7, A549, Hela, and KB cells with IC50 values of 2.02, 0.82, 1.14, and 1.10 μM, respectively.22 Compound 3 showed antiproliferative activity (IC50 = 0.5 μM) against NIAS-SL64 cells, whereas beauvericin exhibited moderate inhibitory activity (IC50 = 6.2 μM) against NIAS-SL64 cells. Compounds 2 and 4 did not show antiproliferative activity against NIAS-SL64 cells (no inhibition at 50 μM).
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured with a P-1030 polarimeter (JASCO, Tokyo, Japan). UV spectra were obtained using a U-2800 spectrophotometer (Hitachi High-Tech, Tokyo, Japan). IR spectra were obtained using an FT/IR4100 Fourier transform infrared spectrometer (JASCO). NMR spectra were recorded using a JNM-ECA600 instrument (JEOL, Tokyo, Japan) with TMS as an internal standard. HRESIMS spectra were measured using an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Fungus Isolation and Taxonomy. The new asteltoxin-producing strain 8-H-28 was isolated from the fruiting body of E. capitata, which was collected at Hanno, Saitama Prefecture, Japan. Genomic DNA was isolated from the hyphae of the fungus grown on potato dextrose agar (Becton, Dickinson and Company, Sparks, MD, USA). Molecular phylogenetic analysis resulted in two possibilities: Pochonia bulbillosa or Pochonia gonioides as the asteltoxin-producing fungus. The strain has falcate conidia that are characteristic of P. bulbillosa23 (see graphical abstract: hyphae and conidia were stained with phenol cotton blue). The isolated strain 8-H-28 was identified as P. bulbillosa based on its phylogenetic and morphological characteristics. The nucleotide sequences of the ITS and 28S regions of P. bulbillosa 8-H-28 were deposited in the DDBJ/EMBL/GenBank databases with the accession numbers LC010959 for ITS and LC010960 for 28S. The ITS and 28S gene sequences are 97.3% and 100% identical respectively between P. bulbillosa 8-H-28 and another P. bulbillosa (NBRC 102299: accession no. AB378554) and also 97.2% and 100% identical respectively between P. bulbillosa 8-H-28 and another P. bulbillosa (NBRC 102305: accession no. AB378555) based on the DDBJ/ EMBL/GenBank databases. Cell Culture. NIAS-SL64 cells17 derived from the fat body of the fifth instar larvae of the common cutworm Spodoptera litura were grown at 25 °C in insect-specific cell culture medium MGM-45024 supplemented with 10% fetal bovine serum. Cytotoxicity Assay. Cell viability was measured by the MTS [3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega)] colorimetric method. NIAS-SL64 cells
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ASSOCIATED CONTENT
S Supporting Information *
1
H and 13C NMR, DEPT, 1H−1H COSY, 1H−13C HMQC, H−13C HMBC, ROE spectra for compounds 2−4. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/np500676j. 1
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81-3-3441-4173. Fax: +81-3-3441-7589. E-mail:
[email protected] (H. Adachi). Notes
The authors declare no competing financial interest. D
DOI: 10.1021/np500676j J. Nat. Prod. XXXX, XXX, XXX−XXX
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Note
ACKNOWLEDGMENTS The authors thank Prof. S. Natori for helpful discussions. This research was supported by the Intramural Research Project of the Microbial Chemistry Research Foundation (MCRF).
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REFERENCES
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